Homozygosity for one of several amino acid substitutions or truncation mutations affecting the C-terminal domain of NOD2/CARD15 is associated with a >30-fold increased risk of developing Crohn's disease (CD) in Western countries. CD-associated mutations of NOD2 have been reported to abolish its ability to recognize the bacterial cell wall component, muramyl dipeptide (MDP), and activate NF-KB signaling in transfected epithelial cells, yet NF-KB is strongly activated in NOD2-expressing intestinal macrophages in CD patients. To resolve this quandary and elucidate the underlying mechanisms, we generated a novel genetic mouse model in which the endogenous Nod2 gene was replaced by a mutant gene, Nod2(2939/C), which codes for a truncated NOD2 protein equivalent to the 1007fs mutation most commonly found in CD patients. Our initial studies show that mice homozygous for this mutation exhibit greater acute inflammation in the DSS-induced model of colitis, and their macrophages are more responsive to MDP, displaying enhanced activation of NFKB signaling and release of mature IL-1beta. These data suggest the overall hypothesis that the mutant Nod2(2939/C) allele codes for a protein with a gain-of-function that serves as a "microbial supersensor". We propose to undertake a series of studies to determine the effects of the NOD2 variant on intestinal inflammation and host defense, and the mechanisms underlying these effects. In Aim 1, we will define the effects of the NOD2 variant on the severity and course of experimentally-induced murine colitis and on immune defense against enteric pathogens. In Aim 2, we will begin to elucidate the mechanisms by which the NOD2 variant mediates its physiological effects on intestinal inflammation and host defense, with a focus on the responsible cell types and the roles of IL-1beta and RICK/Rip2, and the intestinal microbiota in these processes. Aim 3 will utilize cultured macrophages from Nod2(2939/C) mice as a reductionist yet relevant in vitro model to determine the effect of the NOD2 variant on cell signaling and gene expression responses to microbial products, and will define the mechanisms underlying the increased release of mature IL-1beta in cells with mutant NOD2. Together, the proposed studies are expected to yield important new insights into the physiological functions of mutant NOD2 as a mechanistic basis for designing new strategies in the treatment and prevention of CD.